The microvillar enzyme, aminooligopeptidase (AOP), a large glycoprotein present in high concentrations in the intestinal enterocyte, plays an essential role in the surface digestion of nutrient oligopeptides produced by luminal action of pancreatic proteases. The activity of this intestinal brush border hydrolase is low at birth and then steadily increases reaching a maximum towards the end of the first month of life. In the adult, regulation of this microvillar enzyme has been examined in intraintestinal perfusion studies in the rat model using the specific tetrapeptide substrate, Gly-Leu-Gly-Gly. Short-term (30-minute) perfusion of this oligopeptide induces a doubling in the incorporation of [3H]-L-leucine into the AOP associated with intracellular membranes. The molecular events controlling both the developmental expression of this enzyme during ontogeny, as well as its regulation by nutrient oligopeptides are poorly understood. In the proposed work, a rat small intestinal cDNA library which has been constructed in the lambdagt11 expression vector will be screened to identify a specific cDNA probe for AOP. This will be carried out using either polyvalent monospecific rabbit anti-rat AOP antibody or 32P-labeled DNA probes generated by the polymerase chain reaction. Sequencing of the AOP cDNA will then be performed and the amino acid sequence of the enzyme deduced. The molecular events controlling the developmental expression of this enzyme during ontogeny will be studied next during fetal and neonatal life, using the AOP cDNA probe to measure both specific mRNA levels and AOP gene transcription in isolated nuclei at different stages of development. Similar experiments measuring mRNA levels and gene transcription utilizing the specific AOP cDNA probe will also be carried out to further define the effects of short-term intraintestinal perfusion of the specific tetrapeptide substrate, Gly-Leu-Gly-Gly, in the intact adult rat. Finally, the structural organization of the AOP gene will be determined by sequencing of the gene and comparison of this sequence to that of the full-length AOP cDNA. This information will be critical for future investigations of the specific DNA sequences involved in the regulation of AOP gene expression. The proposed research should provide an in depth understanding of the molecular regulation of this essential brush border enzyme.